V-ATPase/TORC1-mediated ATFS-1 translation directs mitochondrial UPR activation in C. elegans

Stressed mitochondria activate the mitochondrial unfolded protein response (UPRmt) to improve organismal health. Li et al. show that increased ATFS-1 translation, mediated by v-ATPase/TORC1 and involving multiple cellular organelles including lysosomes and ribosomes, plays an essential role in UPRmt activation and mild mitochondrial stress-induced longevity.


Introduction
Mitochondria are essential organelles participating in numerous cellular processes, such as energy harvesting, intermediate metabolism, calcium buffering, apoptosis, and immune response (Mishra and Chan, 2014;Nunnari and Suomalainen, 2012;West and Shadel, 2017). Although mitochondria possess their own genome, most mitochondrial proteins are encoded in the nucleus. Therefore, the expression of the mitochondrial proteome requires tight coordination between the two genomes to adapt to changes in the cellular milieu and extracellular environment (Mottis et al., 2019;Zhu et al., 2022). The mitochondrial unfolded protein response (UPR mt ), a branch of the mitochondrial stress response (MSR), is an adaptive transcriptional response that aims at resolving protein-folding stress by orchestrating the remodeling of gene expression programs after multiple forms of mitochondrial stress (Mottis et al., 2019;Shpilka and Haynes, 2018). In general, the activation of the UPR mt preserves fitness during aging and delays the onset of age-related-diseases (Higuchi-Sanabria et al., 2018;Lima et al., 2022;Sun et al., 2016;Vafai and Mootha, 2012).
Although first described in mammalian cells (Zhao et al., 2002), the UPR mt has been mainly studied in the nematode Caenorhabditis elegans. In C. elegans, the UPR mt is induced when the stress-activated transcription factor-1 (ATFS-1) translocates to the nucleus in response to mitochondrial perturbations . In addition, a number of other transcription factors/co-factors, histone methyltransferases, demethylases, acetyltransferases, and deacetylases work together with ATFS-1 during UPR mt activation (Haynes et al., 2007;Li et al., 2021;Merkwirth et al., 2016;Shao et al., 2020;Tian et al., 2016;Yuan et al., 2020;Zhu et al., 2020). Despite the fact that some evidence implicated the involvement of TORC1 components in UPR mt signaling Haynes et al., 2007;Runkel et al., 2013;Shpilka et al., 2021), these studies either did not explore in detail the molecular mechanisms involved Haynes et al., 2007;Runkel et al., 2013) or primarily focused on the role of TORC1 activity in development-associated UPR mt , which is for mitochondrial network expansion (Shpilka et al., 2021). Thus, the function of TORC1 during mitochondrial stress and the mechanism of how TORC1 mediates UPR mt activation remains to be revealed. In mammalian cells, mitochondrial dysfunction triggers the integrated stress response (ISR; Costa-Mattioli and Walter, 2020;Pakos-Zebrucka et al., 2016), in which the phosphorylation of the eukaryotic translation initiation factor 2α (EIF2α) results in the translation of the ATF4, ATF5, and CHOP transcription factors that jointly coordinate a gene expression program considered the functional equivalent of the UPR mt (Mottis et al., 2019;Shpilka and Haynes, 2018). However, little is known about how the mitochondrial stress signal is transmitted through the cytosol and sensed by these UPR mt transcription factors/co-factors. Furthermore, whether the communication between mitochondria and other cellular organelles, such as the lysosomes and ribosomes, contribute to the activation of the UPR mt remains poorly understood.
Here, we demonstrate that stressed mitochondria increase TORC1 activity through a v-ATPase-and Rheb-dependent mechanism in C. elegans. Activated TORC1 thereby leads to increased translation of the UPR mt transcription factor, ATFS-1, a process mediated by cytosolic ribosomes. The accumulated ATFS-1 protein, which is excluded from mitochondria , then translocates to the nucleus and mediates the induction of a specific panel of UPR mt effector genes. Many of these UPR mt effectors play positive roles in the recovery of mitochondrial function, metabolic reprogramming, and lifespan extension. Collectively, our findings reveal a pivotal role of v-ATPase-TORC1-ATFS-1 signaling in UPR mt activation and mild mitochondrial stress-induced longevity. Furthermore, the current study highlights that cytosolic relay of the mitochondrial stress signal from mitochondria to the nucleus also relies on the tight coordination of multiple cellular organelles, including lysosomes and ribosomes.
Similar to the effects of v-ATPase RNAi, suppression of worm TORC1 activity by either RNAi of let-363 (the worm ortholog of human mTOR) and rheb-1 (the worm ortholog of mTORC1 upstream activator, Rheb, Ras homolog enriched in brain; Inoki et al., 2003), or by applying the TORC1 catalytic inhibitor Torin1 (Thoreen et al., 2009) attenuated UPR mt induced by cco-1 or mrps-5 knockdown (Fig. 3 C and Fig. S2 C). Notably, we found that ATFS-1 accumulation and RSKS-1 phosphorylation in response to cco-1 RNAi were abrogated by RNAi of let-363 or rheb-1 and by Torin1 treatment, while EIF-2α phosphorylation and atfs-1 mRNA expression were barely affected (Fig. 3, D-G). Consistent with the importance of lysosomes in TORC1 activation (Lawrence and Zoncu, 2019), the lysosomal acidification inhibitor CQ attenuated the induction of UPR mt genes, TORC1 activity (Fedele and Proud, 2020;Jewell et al., 2015), and the accumulation of ATFS-1 in a dose-dependent manner (Fig. S2, D and E), confirming that the intact lysosomal function/pH is essential for TORC1 and UPR mt activation. Moreover, let-363 or rheb-1 RNAi did not affect the activation of the UPR ER or UPR CYT (Fig. S2, F and G). Intriguingly, knockout of raga-1, the sole worm ortholog of the evolutionarily conserved ras-related GTPase RagA and RagB that are essential for the activation of the mTORC1 by exogenous amino acids (Sancak et al., 2008;Schreiber et al., 2010), lead to even more robust induction of UPR mt genes in response to cco-1 RNAi (Fig. S2 H), suggesting that mitochondrial stress probably represents a unique intrinsic signal for TORC1 activation independent of the Rag GTPase (Hesketh et al., 2020).
The protein level of ATFS-1 has been reported to be mainly controlled by the mitochondrial Lon protease homolog (LONP-1; Nargund et al., 2012;Shpilka et al., 2021;Yang et al., 2022), which mediates the degradation of ATFS-1 in the mitochondrial matrix so that little ATFS-1 is available for the transcriptional activation of the UPR mt in unstressed worms. Indeed, lonp-1 RNAi led to the upregulation of ATFS-1 protein without affecting its mRNA level and UPR mt activity (Fig. 3, H and I). However, more accumulation of ATFS-1 protein in response to cco-1 RNAi was detected even in the background of 50% of lonp-1 RNAi (Fig. 3 H), a condition when lonp-1 transcript was firmly knocked down (Fig. 3 I). As expected, lonp-1 RNAi only increased the accumulation of mitochondrial-localized ATFS-1 (the lower band, produced after the cleavage of its mitochondrial targeting sequence; Nargund et al., 2012), while cco-1 RNAi led to the accumulation of both the mitochondrial-localized and unprocessed forms of ATFS-1 (Fig. 3 H). Importantly, RNAi of vha-1, vha-4, vha-16, and vha-19 blocked the accumulation of ATFS-1 induced by mitochondrial stress when lonp-1 was silenced (Fig. S2 I). Likewise, other mitochondrial stress inducers, such as mrps-5, spg-7, cts-1, and dlst-1 RNAi, also increased ATFS-1 expression and RSKS-1 phosphorylation in the background of lonp-1 RNAi, and these responses were furthermore abolished by vha-1 RNAi (Fig. 3 J). These results suggest that v-ATPase regulates ATFS-1 protein expression upstream of LONP-1 and through a previously uncharacterized LONP-1-independent mechanism.
Knockdown of ribosomal subunits blocks the UPR mt and ATFS-1 translation To test whether increased accumulation of ATFS-1 protein upon mitochondrial stress is caused either by the direct increase of atfs-1 translation or by the suppression of other yet unknown ATFS-1 degradation pathways (e.g., through a ULP-4-mediated SUMOylation mechanism [Gao et al., 2019]), we first knocked down a set of ribosomal subunits, including the small (rps-8 and rps-10) and large ones (rpl-14, rpl-25.1, rpl-27, rpl-36 and rpl-43) in the UPR mt reporter hsp-6p::gfp strain. RNAi of each individual ribosomal subunit remarkably blocked cco-1 or mrps-5 RNAiinduced UPR mt activation, while tunicamycin-induced UPR ER was only partially attenuated, and the UPR CYT /heat shock response was not affected (Fig. 4 A and Fig. S3, A-C). In support of these results, paraquat-induced UPR mt was also reported to rely on multiple ribosomal subunits in a previous screen study (Runkel et al., 2013). Importantly, RNAi of rps-8, rps-10, rpl-27, and rpl-36 almost completely blocked ATFS-1 expression in the atfs-1p::atfs-1::flag::gfp strain upon mitochondrial stress (Fig. 4 B), while, in apparent contrast, the mRNA level of atfs-1 was even upregulated in response to their knockdown (Fig. 4 C). Next, we took advantage of the atfs-1 reporter strain atfs-1p::H1-wCherry (Murray et al., 2012), which was constructed such that the expression of the Histone-mCherry reporter protein is under the strict control of the upstream intergenic sequences (including both the promoter and 59-UTR regions) of atfs-1. Thus, the expression/translation of this Histone-mCherry is controlled in a similar fashion as that of the endogenous ATFS-1 protein. In addition, since the translated Histone-mCherry is not degraded by LONP-1 or other ATFS-1-specific-targeting enzymes, the atfs-1p::Histone-wCherry transgenic strain is therefore an ideal system to study ATFS-1 translation regulation, independent of its degradation. Similar to the results acquired with the atfs-1p::atfs-1:: flag::gfp worms (Fig. 3, A and D; and Fig. 4 B), we found that cco-1 RNAi led to a robust upregulation of the Histone-mCherry reporter protein in the atfs-1p::H1-wCherry worms, which was almost completely blocked by RNAi targeting let-363, rheb-1, v-ATPase, or the ribosomal subunits (Fig. 4, D-F). Finally, we applied polysome profiling whereby free ribosomal subunits, monosomes (mRNA with one ribosome associated), and polysomes (mRNA with two or more ribosomes associated, the highly translated ribosome-mRNA fraction), were separated over a sucrose density gradient and quantified by optical density. Similar to the results as shown for mrps-5 RNAi treatment (Molenaars et al., 2020), cco-1 RNAi led to a shift from polysomes to monosomes (Fig. 4 G), confirming an adaptive cytosolic translation reduction in response to mitochondrial stress Molenaars et al., 2020;Suhm et al., 2018). Importantly, increased polysomal mRNA of atfs-1, as well as that of UPR mt targets hsp-6 and gpd-2, was detected in response to cco-1 RNAi, a phenomenon which is strongly attenuated with vha-1 RNAi co-treatment (Fig. 4 H). Of note, the overall cellular mRNA level of atfs-1 was rather higher on vha-1 RNAi (Fig. 3 E). In contrast, the polysomal mRNA of other transcription factors or typical reporter genes (e.g., hsp-4, dve-1, xbp-1s, hsf-1, and daf-16) involved in different stress responses was not affected by mitochondrial stress and was either unchanged or even upregulated after the co-treatment of vha- 1 RNAi (Fig. 4 H and Fig. S3 D). Of note, neither vha-1 RNAi nor mitochondrial stress altered the overall total and polysomal mRNA levels of rsks-1 (Fig. S3, E and F). Together, these results suggest that increased translation of atfs-1, mediated by v-AT-Pase/TORC1 and cytosolic ribosomes, is a key mechanism that leads to the accumulation of ATFS-1 protein for UPR mt activation in response to mitochondrial stress.
Mitochondrial stress-induced ATFS-1 is independent of the GCN-2/PEK-1 signaling In mammals, phosphorylation of EIF2α by four dedicated kinases (GCN2, PERK, HRI, and PKR) serves to attenuate the general cytosolic translation in response to a variety of intra-and extracellular stresses (e.g., amino-acid starvation, viral infection, oxidative and unfolded protein stress), and meanwhile stimulates the translation of ATF4, ATF5, and CHOP, the mammalian functional orthologs of ATFS-1, to activate the ISR (Costa-Mattioli and Walter, 2020;Pakos-Zebrucka et al., 2016;Quiros et al., 2017). We thus questioned whether a similar mechanism also exists in C. elegans. Surprisingly, RNAi of gcn-2 and/or pek-1, which encode the only two known corresponding worm EIF-2α kinases, GCN-2 and PEK-1 Shen et al., 2001), failed to block cco-1 RNAi-induced UPR mt in hsp-6p::gfp worms (Fig. 5 A). Meanwhile, EIF-2α phosphorylation was attenuated by either gcn-2 or pek-1 RNAi, in both basal and mitochondrial stress conditions (Fig. 5 B). Moreover, although only GCN-2 is required for EIF-2α phosphorylation in the clk-1(qm30) mitochondrial mutant Lakowski and Hekimi, 1996), less of EIF-2α phosphorylation was detected with the cosilencing of both gcn-2 and pek-1 upon cco-1 RNAi (Fig. 5 B). In line with the results acquired in hsp-6p::gfp worms (Fig. 5 A), mitochondrial stress-induced upregulation of ATFS-1 was not affected by either gcn-2, pek-1, or eif-2α RNAi (Fig. 5, B and C). Furthermore, comparable levels of RSKS-1 phosphorylation and UPR mt transcript induction upon cco-1 RNAi were found in the gcn-2 or pek-1 knockout worm mutants, even in conditions with full suppression of EIF-2α phosphorylation, as compared with that in WT (N2) worms (Fig. 5, D-F). Likewise, activation of the UPR mt was also not affected in autophagy-defective mutants (Fig. S4). Collectively, these results suggest that increased expression of ATFS-1 in response to mitochondrial stress is independent of the GCN-2/PEK-1 signaling as well as the autophagic process per se.

A crucial role of v-ATPase and ribosomal subunits in mitochondrial surveillance
To explore the functions of v-ATPase and ribosomal subunits in mitochondrial homeostasis and adaptations upon stresses, we raised WT and the mitochondrial respiration mutants isp-1(qm150) and clk-1(qm30) (Feng et al., 2001;Lakowski and Hekimi, 1996), and on control, vha-1, vha-4, vha-16, vha-19, rps-8, rps-10, rpl-27, or rpl-36 RNAi bacteria. Compared to C. elegans fed with control RNAi, RNAi targeting v-ATPase or ribosomal subunits led to severe synthetic growth defects of the mitochondrial stressed mutants, whereas the development of WT worms was only slightly delayed ( Fig. 6 A and Fig. S5 A). Similar results were also found in worms fed with vha-1 RNAi and/or cco-1 RNAi ( Fig. S5 B). Thus, mitochondrial respiration mutants heavily rely on v-ATPase and ribosomal subunits to maintain growth. We then questioned whether v-ATPase and ribosomal subunits also contribute to mild mitochondrial stress-induced lifespan extension in C. elegans (Durieux et al., 2011;Houtkooper et al., 2013). In line with an essential role of the v-ATPase subunits in the UPR mt (Fig. 1, A-C), RNAi of vha-1, vha-4, vha-16, and vha-19 strongly attenuated the lifespan extension induced by cco-1 or mrps-5 RNAi (Fig. 6, B and C). Consistently, the silencing of ribosomal subunits including rps-8, rps-10, rpl-27, and rpl-36 also blunted the lifespan extension induced by cco-1 RNAi (Fig. 6 D). Thus, v-ATPase and ribosomal components play a crucial role in mitochondrial surveillance and regulate the longevity induced by mild mitochondrial stress in C. elegans.

Discussion
The UPR mt was initially defined as a transcriptional response triggered by the presence or accumulation of unfolded proteins/ peptides from mitochondria (Shpilka and Haynes, 2018;Zhao et al., 2002). In C. elegans, almost all the well-characterized UPR mt regulators, such as ATFS-1   (Zhu et al., 2020), and CBP-1 , are localized or translocated in the nucleus upon mitochondrial stress. However, how the mitochondrial stress signal is sensed in the cytosol and relayed to these UPR mt regulators is only partially elucidated. Moreover, whether other protein complexes and organelles, such as the lysosomes and ribosomes, also play a role in the UPR mt activation process remains elusive.
Here, we demonstrated that the mitochondrial stress is transduced through a v-ATPase-TORC1-ATFS-1 signaling pathway in the cytosol involving distinct organelles (Fig. 7). In this signaling network, stressed mitochondria increase TORC1 activity through a v-ATPase-and Rheb-dependent mechanism. Activated TORC1 thereby leads to increased translation of the UPR mt transcription factor, ATFS-1, which is dependent on the cytosolic ribosomes ( Fig. 7; indicated by mechanism 1). The accumulated ATFS-1 protein then translocates to the nucleus and mediates the induction of a specific panel of UPR mt effector genes. Many of these UPR mt effectors play positive roles in the recovery of mitochondrial function, metabolic reprogramming, and lifespan extension. Importantly, genetic or pharmacological disruption of any components in this pathway robustly suppressed the UPR mt , but not other similar stress responses, such as UPR ER and UPR CYT . Our work thus reveals that in addition to the attenuated mitochondrial import of ATFS-1 in response to mitochondrial stress Fig. 7; indicated by mechanism 2), v-ATPase/TORC1-mediated upregulation of ATFS-1 translation is also essential to ensure that enough ATFS-1 is translocated to the nucleus for UPR mt activation. Of note, v-ATPase/TORC1-mediated translation of ATFS-1 seems to be a prerequisite step for the increased accumulation and the subsequent nuclear localization of ATFS-1 for UPR mt activation during mitochondrial stress, as evidenced by the GFP-ATFS-1 nuclear localization results (Fig. 2 I), supporting that the two mechanisms likely act as a whole in MSR.
Consistent with a central role of TORC1 signaling in the UPR mt , TORC1 and RSKS-1 were reported to be indispensable for the increased UPR mt activity to support mitochondrial network expansion during development, a condition when TORC1 activity is already known to be active (Shpilka et al., 2021). How the TORC1 activity is activated in response to mitochondrial stress remains an important direction for future work. One possibility is that the unfolded proteins/peptides produced upon mitochondrial stress could somehow be transported from the mitochondria to the lysosomes and thereby digested to amino acids within the lysosomes, which could then lead to TORC1 activation at the lysosomal surface (Lawrence and Zoncu, 2019;Shimobayashi and Hall, 2014;Wolfson and Sabatini, 2017;Zoncu et al., 2011). We found that the cco-1 RNAi-induced UPR mt activation is independent of raga-1 (Fig. S2 H), suggesting that mitochondrial stress likely represents a unique intrinsic signal for TORC1 activation by lysosome-derived amino acids (Hesketh et al., 2020), which apparently differs from what is observed during development or upon stimulation with exogenous amino acids (Sancak et al., 2008;Schreiber et al., 2010). In support of this model, a Rab5-mediated mitochondrion-endosomelysosome pathway functions in mitochondrial quality control and is activated upon mitochondrial dysfunction, independent of the autophagic process (Hammerling et al., 2017;Sugiura et al., 2014). More mitochondrial proteins/peptides were also detected in Rab5-positive endosomes in response to mitochondrial stress (Hammerling et al., 2020). Of note, stressed mitochondria might also directly communicate with lysosomes via mitochondrialysosome membrane contact sites (Wong et al., 2019). Finally, v-ATPase has been also found to participate in endosomal membrane fusion processes (Peters et al., 2001), and it may thereby facilitate the transportation of mitochondria-derived unfolded proteins/peptides to the lysosomes, together with Rab5 and other cofactors.
Table S1), indicating that v-ATPase functions in maintaining basal UPR mt activity as well. Nevertheless, the difference between basal and stress conditions is somehow artificial, especially considering that cells and organisms are constantly exposed to various intra-and extracellular cues, and different wild C. elegans strains differ at the level of UPR mt activity under basal conditions (Yin et al., 2017).

Li et al.
Journal of Cell Biology v-ATPase/TORC1-driven MSR pathway could have therapeutic applications against mitochondria-associated diseases, metabolic disorders, as well as normal aging in other organisms.
Limitations of the study While our current study reveals an indispensable role of v-AT-Pase/TORC1-mediated ATFS-1 translation in UPR mt activation and mitochondrial stress-associated lifespan extension in C. elegans, several limitations exist. First, how the lysosomes or the v-ATPase/TORC1 complex senses the mitochondrial stress is not well addressed in the current work. To fill this gap, highresolution imaging and systematic proteomic analysis of the changes in the content of endosomal and lysosomal vesicles in response to mitochondrial stress would be required in the future. Second, we did not manage to find a way to quantitate and compare the magnitude of the effects of v-ATPase/TORC1mediated ATFS-1 translation versus the suppression of mitochondrial import as nonmutually exclusive mechanisms for UPR mt activation. Third, despite that the decreased expression of ATFS-1 protein is sufficient to explain the reduced activation of UPR mt upon v-ATPase/TORC1 inhibition in C. elegans, we cannot exclude that TORC1 or other enzymes, such as CBP-1 , could still contribute to the UPR mt through posttranslational modifications (e.g., phosphorylation, acetylation) of ATFS-1. Finally, it has been extensively reported that TORC1 signaling inhibition extends the lifespan in multiple animal models (Saxton and Sabatini, 2017;Shimobayashi and Hall, 2014). However, in our current study as well as in other published work (Shpilka et al., 2021), TORC1 activity seems also to be essential for the mitochondrial stress response, which generally extends the lifespan as well. Thus, how these two intertwined processes coordinate to determine overall organismal health and lifespan still requires further investigation.

C. elegans strains
The Bristol strain (N2) was used as the wild-type strain. RNA interference and drug treatment E. coli strain HT115(DE3; RRID:WB-STRAIN:WBStrain00041080) was obtained from the CGC, transformed with the empty vector L4440, and used as the RNAi control. RNAi clones were obtained from either the Ahringer or Vidal library (Kamath et al., 2003;Figure 7. Model for v-ATPase/TORC1-mediated regulation of ATFS-1 translation and UPR mt activation in C. elegans. When mitochondria are stressed in response to various intracellular or extracellular stimuli, the activity of TORC1 is increased through a v-ATPase-and Rheb-dependent mechanism, which could be blocked by both inhibitors of v-ATPase, Bafilomycin A1 (BafA1), and Concanamycin A (ConA); the lysosomal acidification inhibitor, chloroquine (CQ); as well as the TORC1 inhibitor, Torin1. Activated TORC1 thereby stimulates cytosolic ribosomes to translate the UPR mt transcription factor ATFS-1 (mechanism 1). Under the basal nonstressed condition, ATFS-1 is transported and degraded in the mitochondria; during mitochondrial stress, in addition to increased ATFS-1 translation, mitochondrial import efficiency is also decreased (mechanism 2; Nargund et al., 2012). Both mechanisms together result in nuclear accumulation of ATFS-1, where it induces a diverse panel of UPR mt effector genes. Many of these UPR mt effectors play positive roles in mitochondrial function recovery, metabolic reprogramming, and lifespan extension. Rual et al., 2004) and further verified by sequencing or qRT-PCR. The accession codes for vha-1 RNAi clones were 10008-A6 (vha-1_RNAi_1) from the Vidal library and III-5A20 (vha-1_ RNAi_2) from the Ahringer library. Double RNAi experiments were performed by mixing bacterial cultures normalized to their optical densities (OD 600 ) before seeding. For treatment of worms with compounds, stock solutions of Tunicamycin (Cat. T7765; Sigma-Aldrich), Concanamycin A (Cat. C9705; Sigma-Aldrich), Bafilomycin A1 (Cat. S1413; Selleckchem), or chloroquine (CQ, Cat. C6628; Sigma-Aldrich) were added to the NGM with final concentrations, as indicated in the figure legends, just before pouring the plates.
UPR mt induction and imaging in C. elegans For RNAi-induced UPR mt , RNAi bacteria were cultured in lysogeny broth (LB) medium containing 100 μg/ml ampicillin at 37°C overnight. The bacteria were then seeded onto NGM plates containing 2 mM IPTG and 25 mg/ml carbenicillin. L4/young adult worms were picked onto the RNAi bacteria-seeded plates and cultured at 20°C until their progenies reached the young adult stage. A total of 5-10 progenies were then randomly picked and aligned in 10 mM tetramisole (Cat. T1512; Sigma-Aldrich) droplets on NGM plates. Fluorescent images, with the same exposure time for each condition within each of the experiments, were acquired using a Nikon SMZ1000 microscope. All images are compared relatively only to each negative and positive controls in the same batch of the experiment. For compound-induced UPR mt , antimycin A (Cat. A8674; Sigma-Aldrich) with a final concentration of 2.5 μM or Dox (Cat. D9891; Sigma-Aldrich) with a final concentration of 30 μg/ml was added into the NGM just before pouring the plates. For imaging GFPtagged ATFS-1, atfs-1p::atfs-1::flag::gfp (OP675) worms were mounted in 10 mM tetramisole (Cat. T1512; Sigma-Aldrich) on 2% agarose pads. The most proximal two intestinal cells in each worm were assessed with a Zeiss LSM 700 confocal microscope. At least 20 nuclei were analyzed for each condition. All images were acquired at room temperature with the software provided with the corresponding microscope.

Lifespan experiments
Lifespan experiments were performed at 20°C as described previously (Houtkooper et al., 2013). Briefly, 80-100 worms were used per condition and scored every other day, and those that disappeared or exploded at the vulva were censored. Worms were transferred to fresh plates every week. All RNAi treatments for lifespan started at the maternal L4 stage.

RNA extraction and RNA-seq analysis
For worm samples, synchronized worm eggs were seeded onto NGM plates and cultured at 20°C until the worms reached L4/ young adult stage. Worms were then harvested with M9 buffer and snap-frozen in liquid nitrogen. For RNA extraction, 1 ml of TriPure Isolation Reagent (Cat. 11667165001; Roche) was added to each sample tube. The worms were then frozen with liquid nitrogen and thawed in a water bath quickly eight times to rupture cell membranes. Total RNA was then extracted using a column-based kit (Cat. 740955.250;. All RNAseq was performed by BGI with the BGISEQ-500 platform. For RNA-seq results, the raw data were filtered by removing adaptor sequences, contamination, and low-quality (phred quality <20) reads. Qualified reads were then mapped to either the worm "Caenorhabditis_elegans.WBcel235.89" with STAR aligner version 2.6.0a. Reads were counted using htseq-count version 0.10.0 using these flags: -f bam -r pos -s no -m union -t exon -i gene_id. Differential expression of genes was calculated by Limma-Voom. The genes with a Benjamini-Hochberg adjusted P value <0.05 were defined as statistically significant. Genes whose expressions were significantly upregulated with log 2 FC > 0.393 (the fold change for hsp-6, which encodes the UPR mt maker protein HSP-6) in cco-1 RNAi condition and were then downregulated by more than 25% of the log 2 FC after vha-1/-4/-16/-19 or atfs-1 RNAi co-treatment, compared with the log 2 FC of cco-1 RNAi condition, were considered as VHA-or ATFS-1-dependent. Functional clustering was conducted using the DAVID (Database for Annotation, Visualization, and Integrated Discovery) database (Huang et al., 2009). Heat-maps were generated by Morpheus (https://software.broadinstitute.org/morpheus). UpSet plot was generated by Intervene (https://asntech.shinyapps.io/intervene/; Khan and Mathelier, 2017).

Quantitative RT-PCR (qRT-PCR)
Worms were harvested and total RNA was extracted as described above for RNA-seq. cDNA was synthesized using the Reverse Transcription Kit (Cat. 205314; Qiagen). qRT-PCR was conducted with the LightCycler 480 SYBR Green I Master kit (Cat. 04887352001; Roche). Primers used for qRT-PCR are listed in Table S2. Primers for worm pmp-3 were used as the normalization control.

Statistical analysis
No statistical methods were used to predetermine the sample size. Investigators were not blinded to allocation during experiments and outcome assessment. All experiments, except for the RNA-seq, were repeated at least twice, and similar results were acquired. All statistical analyses were performed using Graphpad Prism 8 software. Data distribution was assumed to be normal, but this was not formally tested. Differences between the two groups were assessed using two-tailed unpaired Student's t tests. Analysis of variance (ANOVA) followed by Tukey post-hoc test (one-way ANOVA for comparisons between groups, and two-way ANOVA for comparisons of magnitude of changes between different groups from different treatments or cell lines) was used when comparing more than two groups. Survival analyses were performed using the Kaplan-Meier method, and the significance of differences between survival curves was calculated using the log-rank (Mantel-Cox) method. Fig. S1 shows the impact of vha-1, vha-4, vha-16, or vha-19 RNAi in different stress responses and gene expression in C. elegans. Fig. S2 shows the impact of mitochondrial stress, ER stress, and TORC1 signaling regulators in gene expression and stress responses. Fig. S3 shows the impact of ribosomal subunit RNAi in stress responses in C. elegans. Fig. S4 shows that autophagy-defective mutants have normal activation of the UPR mt in response to mitochondrial stress. Fig. S5 shows the key role of ribosomal subunits and VHA-1 in mitochondrial surveillance. Table S1 shows the RNA-seq results of worms fed with RNAi targeting v-ATPase subunits and/or cco-1 RNAi. Table S2 shows the list of primers used for qRT-PCR in this study.